We have demonstrated that a number of wild E. coli strains show few nucleotide differences from E. coli strain K12 in the trpCBA region, and that the differences are clustered with respect to strains, to codon position, and to location within the gene. For 25-40 strains of each species, we now plan to sequence a DNA fragment about 500 bp in length from each of several regions of the E. coli chromosome (translated and untranslated), and from each of two regions of the B. subtilis chromosome. Where considerable variation is revealed, longer fragments will be sequenced. This research will show whether general conclusions on intraspecific phylogeny can be drawn from one region alone, and whether B. subtilis is sufficiently different in its population dynamics (recombination rate, rate of spread) to evince a different pattern of variation. Chromosomal DNA will be cut, ligated to pBR322, and transformed into recipient E. coli strains with appropriate properties (lacking restriction endonucleases and lacking the property conferred by the particular DNA to be selected). After amplification, the DNA will be fractionated and sequenced. Further information on the process of spontaneous mutation is also anticipated, given the occasionally dramatic clustering observed to date; alternatively, a selective basis for the clustered substitutions may reveal further functional significance of local DNA or RNA structure.